Corneal endothelial cell marker

ABSTRACT

A molecular marker expressed specifically in corneal endothelial cells, and a method for producing one or more corneal endothelial cells using the marker and a method for evaluating one or more corneal endothelial cells using the marker are provided. At least one molecule selected from the group consisting of ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, and CLRN1 is used as a marker specific to corneal endothelial cells.

TECHNICAL FIELD

The present invention relates to a molecular marker expressedspecifically in corneal endothelial cells, a method for producingcorneal endothelial cells using the marker and a method for evaluatingcorneal endothelial cells using the marker.

BACKGROUND ART

The cornea is the transparent front part of the eyeball. From theanterior, the cornea has a corneal epithelium, a Bowman's membrane, acorneal stroma, a Descemet's membrane, and a corneal endothelium. Thecorneal epithelium is present at the outermost part of the cornea, andfunctions as a barrier to protect the cornea from foreign matter, suchas dust and bacteria. The Bowman's membrane is thought to function as abase for the corneal epithelium. The corneal stroma is the thickestlayer among the three layers (i.e., corneal epithelium, corneal stroma,and corneal endothelium), and maintains the strength of the cornea. TheDescemet's membrane is located beneath the corneal stroma, and connectsthe corneal stroma with the corneal endothelium. The corneal endotheliumis a monolayer in which hexagonal corneal endothelial cells areregularly arranged like cobblestones; it plays a critical role inmaintaining the transparency of the cornea by preventing, by its barrierand pumping functions, the moisture in the anterior chamber frompenetrating into the corneal stroma, while maintaining the fluid balanceby draining the moisture in the cornea out to the anterior chamber.

Loss of a significant number of corneal endothelial cells due to celldamage caused by genetic or external factors impairs the functionsdescribed above, leading to corneal edema. Severe corneal endothelialdysfunction compromises the maintenance of the transparency of thecornea. If bullous keratopathy develops, marked visual loss may result.

Corneal endothelial cells have a very limited capacity for proliferationin the human body. The most effective, fundamental method to treatsevere corneal endothelial dysfunction at the moment is cornealtransplantation. In fact, bullous keratopathy is the disease for whichcorneal transplantation has been most often performed. Althoughfull-thickness cornea transplants have, in the past, been performed forcorneal endothelial dysfunction, there are problems such as a chronicdonor shortage and transplant rejection. To minimize rejection, atechnique has been developed that transplants only a partial tissuecontaining a corneal endothelium to an eye with a disorder (Descemet'sStripping Automated Endothelial Keratoplasty: DSAEK). However, DSAEKalso cannot overcome the donor shortage. Attempts to grow cornealendothelial cells in vitro and use the cells in treatment have also beenmade (Non-patent Literature 1 and Patent Literature 1 to 3). However,repeated passages cause genetic transformation, leading to loss of theirfunctions (Non-patent Literature 2).

To solve the problems described above, a number of studies have beenconducted on induction of corneal endothelial cells from umbilical cordblood-derived mesenchymal stem cells (Non-patent Literature 3), bonemarrow-derived cells (Non-patent Literature 4), iris-derived stem cells(Non-patent Literature 5), corneal stroma-derived stem cells (Non-patentLiterature 6), embryonic stem cells (Non-patent Literature 7), inducedpluripotent stem cells (Patent Literature 4), etc., for the purpose oftransplant. However, regardless of the origin of the cells, thethus-obtained cultured cells contain different types of cells inaddition to corneal endothelial cells. In these studies, therefore, amarker specific to corneal endothelial cells usable to isolate andevaluate corneal endothelial cells (i.e., its target product) isindispensable.

Currently, proteins, such as ZO-1 (Non-patent Literature 8),Na⁺—K⁺-ATPase (Non-patent Literature 9), and N-cadherin (Non-patentLiterature 10), are used as corneal endothelial cell markers. Theseproteins, however, are expressed non-specifically in many other types ofcells (Non-patent Literature 11 to 13). Thus, isolation of cornealendothelial cells using these proteins is not expected to give asatisfactory outcome, in particular given the multilineage potential ofthe stem cells used to induce differentiation into corneal endothelialcells.

CITATION LIST Patent Literature

-   Patent Literature 1: WO2006/092894A1-   Patent Literature 2: WO2011/096593A1-   Patent Literature 3: WO2013/012087A1-   Patent Literature 4: WO2013/051722A1

Non-Patent Literature

-   Non-patent Literature 1: Ide T, Nishida K, Yamato M, Sumide T,    Utsumi M, et al. (2006) Structural characterization of bioengineered    human corneal endothelial cell sheets fabricated on    temperature-responsive culture dishes. Biomaterials 27: 607-614.-   Non-patent Literature 2: Joyce N C (2003) Proliferative capacity of    the corneal endothelium. Prog Retin Eye Res 22: 359-389.-   Non-patent Literature 3: Joyce N C, Harris D L, Markov V, Zhang Z,    Saitta B (2012) Potential of human umbilical cord blood mesenchymal    stem cells to heal damaged corneal endothelium. Mol Vis 18: 547-564.-   Non-patent Literature 4: Shao C, Fu Y, Lu W, Fan X (2011) Bone    marrow-derived endothelial progenitor cells: a promising therapeutic    alternative for corneal endothelial dysfunction. Cells Tissues    Organs 193: 253-263.-   Non-patent Literature 5: Kikuchi M, Hayashi R, Kanakubo S, Ogasawara    A, Yamato M, et al. (2011) Neural crest-derived multipotent cells in    the adult mouse iris stroma. Genes Cells 16: 273-281.-   Non-patent Literature 6: Hatou S, Yoshida S, Higa K, Miyashita H,    Inagaki E, et al. (2013) Functional corneal endothelium derived from    corneal stroma stem cells of neural crest origin by retinoic acid    and Wnt/β-catenin signaling. Stem Cells Dev 22: 828-839.-   Non-patent Literature 7: Zhang, K, Pang, K, Wu, X. (2014) Isolation    and transplantation of corneal endothelial cell-like cells derived    from in-vitro-differentiated human embryonic stem cells. Stem cells    and development 23: 1340-1354.-   Non-patent Literature 8: Barry P A, Petroll W M, Andrews P M,    Cavanagh H D, Jester J V. (1995) The spatial organization of corneal    endothelial cytoskeletal proteins and their relationship to the    apical junctional complex. Invest Ophthalmol Vis Sci 36: 1115-1124.-   Non-patent Literature 9: Zam Z S, Cerda J, Polack F M. (1980)    Isolation of the plasma membrane from corneal endothelial cells.    Invest Ophthalmol Vis Sci 19: 648-652.-   Non-patent Literature 10: Vassilev V S, Mandai M, Yonemura S,    Takeichi M (2012) Loss of N-cadherin from the endothelium causes    stromal edema and epithelial dysgenesis in the mouse cornea. Invest    Ophthalmol Vis Sci 53: 7183-7193.-   Non-patent Literature 11: Howarth A G, Hughes M R, Stevenson B    R (1992) Detection of the tight junction-associated protein ZO-1 in    astrocytes and other nonepithelial cell types. Am J Physiol 262:    C461-469.-   Non-patent Literature 12: Mobasheri A, Avila J, Cozar-Castellano I,    Brownleader M D, Trevan M, et al. (2000) Na⁺, K⁺-ATPase isozyme    diversity; comparative biochemistry and physiological implications    of novel functional interactions. Biosci Rep 20: 51-91.-   Non-patent Literature 13: Tsuchiya B, Sato Y, Kameya T, Okayasu I,    Mukai K (2006) Differential expression of N-cadherin and E-cadherin    in normal human tissues. Arch Histol Cytol. 69: 135-145.

SUMMARY OF INVENTION Technical Problem

In view of the status quo in the art, an object of the present inventionis to provide a molecular marker expressed specifically in cornealendothelial cells, and applied technologies, such as a method forproducing corneal endothelial cells using the marker and a method forevaluating corneal endothelial cells using the marker.

Solution to Problem

The present inventors conducted extensive research to achieve theobject. The inventors selected, from a database that has an exhaustivecoverage of genes expressed in corneal endothelial cells, genes that arehighly expressed in corneal endothelial cells, that encode cellularmembrane proteins, and that exhibit a low level of expression in othertissues. The inventors examined the expression of the genes in cornealendothelial cells and in other tissues to find genes that specificallyexpress in corneal endothelial cells. They also immunostained humancornea sections to detect proteins encoded by these genes, and confirmedtheir specific expression in the corneal endothelium. The inventionrepresented by the following subject matter is provided on the basis ofthese findings.

Item 1.

A method for producing one or more corneal endothelial cells, the methodcomprising the step of sorting, from a cell population comprising one ormore corneal endothelial cells, one or more cells in which at least onemember selected from the group consisting of ZP4, MRGPRX3, GRIP1, GLP1R,HTR1D, and CLRN1 is expressed.

Item 2.

The method according to Item 1, wherein the cell population comprisingone or more corneal endothelial cells is obtained by inducingdifferentiation of stem cells.

Item 3.

The method according to Item 1, wherein the cell population comprisingone or more corneal endothelial cells is obtained by culturing cornealendothelial cells.

Item 4.

The method according to any one of Items 1 to 3, wherein the sortingstep comprises binding one or more antibodies that specificallyrecognize at least one member selected from the group consisting of ZP4,MRGPRX3, GRIP1, GLP1R, HTR1D, and CLRN1 to the cell populationcomprising one or more corneal endothelial cells.

Item 5.

A method for evaluating one or more corneal endothelial cells usingexpression of at least one member selected from the group consisting ofZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, and CLRN1 as an indication.

Item 6.

A marker for detecting one or more corneal endothelial cells, the markercomprising at least one member selected from the group consisting ofZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, and CLRN1.

Item 7.

A kit for detecting one or more corneal endothelial cells, the kitcomprising one or more substances that specifically recognize at leastone member selected from the group consisting of ZP4, MRGPRX3, GRIP1,GLP1R, HTR1D, and CLRN1.

Advantageous Effects of Invention

A molecular marker that is expressed specifically in corneal endothelialcells is provided, and this marker can specifically detect cornealendothelial cells. The expression product of the molecular marker is acell-surface protein. Thus, cells are identified and/or sorted in aviable condition. Specifically, the marker can efficiently providecorneal endothelial cells in a viable condition. Because cornealendothelial cells obtained using this marker are suitable fortransplantation, the use of the marker not only enables treatment ofdiseases that require transplant of corneal endothelial cells, but alsoalleviates the donor shortage in corneal transplantation. The molecularmarker is also usable to accurately evaluate cultured cornealendothelial cells for their suitability for transplantation. The presentinvention is therefore useful in the treatment of various diseasescaused by functional disorder of corneal endothelial cells.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of the measurement of expression levels ofgenes encoding 13 candidate molecules in corneal endothelial cells andin other 22 different tissues. In each chart, abbreviations are used asfollows: CECs: corneal endothelial cells, A-brain: adult brain, F-brain:fetal brain, SC: spinal cord, SG: salivary gland, BM: bone marrow, SM:skeletal muscle, A-liver: adult liver, F-liver: fetal liver, SI: smallintestine. The vertical axis of each chart indicates a gene expressionlevel relative to the expression level of a control.

FIG. 2 shows the results of the measurement of expression levels ofgenes encoding candidate molecules in ocular tissues. The measurementwas performed by quantitative PCR. In each chart, abbreviations are usedas follows: CECs: corneal endothelial cells, CC: cultured cornealendothelial cells, C. stroma: corneal stroma, C. epi: cornealepithelium, Limbus: corneal limbus, Iris pig. epi: iris pigmentepithelial cells, TM: trabecular meshwork, CB: ciliary body,RPE/choroid: retinal pigment epithelium/choroid, and ON: optic nerve.The vertical axis of each chart indicates a gene expression levelrelative to the expression level of a control.

FIG. 3 shows the results of immunostaining of human corneal sections todetect 6 proteins (GRIP1, CLRN1, MRGPRX3, ZP4, GLP1R, and HTR1D).

FIG. 4 shows expression levels of ZP4 before and after the induction ofhuman iPS cells into corneal endothelial cells.

DESCRIPTION OF EMBODIMENTS

1. Method for Producing Corneal Endothelial Cells 1-1. Cell PopulationContaining Corneal Endothelial Cells

A “cell population containing one or more corneal endothelial cells,”which is a starting material for producing one or more cornealendothelial cells, is an assembly of cells including one or more cornealendothelial cells. There is no limitation to the origin and constituentsof the cell population, as long as the population contains one or morecorneal endothelial cells. For example, the cell population containingone or more corneal endothelial cells encompasses a cell populationobtained by artificial differentiation of stem cell(s) and a cellpopulation obtained by culturing corneal endothelial cells isolated froma cornea. Although, in most cases, the cell population containing one ormore corneal endothelial cells contains cells other than cornealendothelial cells, the population may contain only one or more cornealendothelial cells. The origin of the cell population containing one ormore corneal endothelial cells is not particularly limited, but ispreferably a human.

For example, the percentage of corneal endothelial cells present in thecell population containing one or more corneal endothelial cells is 1%or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more,50% or more, 60% or more, 70% or more, or 80% or more, based on thetotal number of cells.

Stem cells for use in preparing the cell population containing one ormore corneal endothelial cells are not particularly limited, as long asthe stem cells can be cultured in vitro and can be differentiated intocorneal endothelial cells. The stem cells for use may be suitablyselected from any stem cells known and hereafter developed in the art.Examples of such stem cells include artificial pluripotent stem cells(induced pluripotent stem cells: iPS cells), embryonic stem cells (EScells), fetal primordial germ cell-derived pluripotent stem cells (EGcells), testis-derived pluripotent stem cells (GS cells), and humansomatic stem cells capable of differentiating into corneal endothelialcells (tissue stem cells).

iPS cells can be obtained by any available technique, for example, byintroducing DNA- or protein-form specific reprogramming factors intosomatic cells. Examples of reprogramming factors include Oct3/4, Sox2,Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28,Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb,Nr5a2, Tbx3, and Glis1. These reprogramming factors may be used singlyor in any combination. Examples of combinations of reprogramming factorsare disclosed in, for example, WO2007/069666, WO2008/118820,WO2009/007852, WO2009/032194, WO2009/058413, and WO2009/057831. The typeof somatic cells is not particularly limited, and somatic cells for useinclude any cells that have been confirmed to produce iPS cells and anycells hereafter reported to produce iPS cells. In an embodiment,examples of preferable somatic cells include fibroblasts and white bloodcells. The somatic cells are preferably derived from humans.

ES cells are obtained by any available technique. For example, ES cellscan be established by removing the inner cell mass from the blastocystof a fertilized egg from a mammal (preferably, a human), and culturingthe inner cell mass on a feeder of fibroblasts. The mammal is notparticularly limited, but is preferably a human. ES cells in passageculture can be maintained using a culture solution containingsubstances, such as a leukemia inhibitory factor (LIF) and/or a basicfibroblast growth factor (bFGF). ES cells can be selected, for example,using expression of gene markers, such as OCT-3/4, NANOG, and ECAD as anindication.

EG cells are pluripotent cells as with ES cells, and are establishedfrom primordial germ cells in the fetal stage. EG cells are establishedby culturing primordial germ cells in the presence of substances, suchas LIF, bFGF, and stem cell factors (Y. Matsui et al. (1992), Cell, 70:841-847; J. L. Resnick et al. (1992), Nature, 359: 550-551).

GS cells are testis-derived pluripotent stem cells, and are the originof sperm formation. These cells can be induced to differentiate into avariety of cell lineages like ES cells. GS cells can be self-renewed ina culture solution containing a glial cell line-derived neurotrophicfactor (GDNF). Repeated passage under the same conditions as those forES cells produces sperm stem cells (M. Kanatsu-Shinohara et al. (2003)Biol. Reprod., 69: 612-616).

Examples of somatic stem cells that can be differentiated into cornealendothelial cells include neural crest stem cells derived from thecorneal stroma (COPs), mesenchymal stem cells, and skin-derivedpluripotent precursor cells (skin-derived precursors: SKPs), with COPsand SKPs being preferable. COPs can be prepared, for example, byremoving the epithelium and endothelium from a cornea, treating thecorneal stroma with a collagenase, and culturing the separated cells ina DMEM/F12 medium containing EGF, FGF2, B27 supplements and LIF. SKPscan be prepared, for example, in accordance with the procedure describedin Nat Cell Biol., 2001, vol. 3, 778-784.

In an embodiment, preferable stem cells are iPS cells. In anotherembodiment, preferable stem cells are neural crest stem cells, and morepreferable stem cells are iPS cell-derived neural crest stem cells andcorneal stroma-derived neural crest stem cells. The neural crest stemcells, which are pluripotent stem cells that have a self-renewalpotential and multilineage potential, are known to migrate out frombehind of the neural tube across the body during the development of avertebrate, and contribute to formation of various tissues. iPS cellscan be induced into neural crest stem cells by a known method in theart, or a method according thereto (e.g., the method described in NatureProtocols, 2010, vol. 5, No. 4, 688-701). The use of neural crest stemcells enables efficient differentiation induction into cornealendothelial cells.

The method for inducing differentiation of the stem cells describedabove into corneal endothelial cells is not particularly limited, andany method known or hereafter developed in the art can be used. Forexample, the differentiation induction can be performed by the methoddisclosed in Patent Literature 4. Specifically, stem cells can bedifferentiated into corneal endothelial cells by culturing the stemcells in a medium that is suitable for culturing stem cells (e.g., MEMmedium) and that contains at least one differentiation-inducing factorselected from the group consisting of a GSK3 inhibitor, retinoic acid,TGFb2, insulin, and a ROCK inhibitor at 30 to 40° C. in 1 to 10% CO₂ forabout several days to one and a half months. The at least onedifferentiation-inducing factor preferably contains a GSK3 inhibitor andretinoic acid, more preferably a GSK3 inhibitor, retinoic acid, and aROCK inhibitor, and still more preferably a GSK3 inhibitor, retinoicacid, a ROCK inhibitor, and insulin.

In the manner described above, a cell population that contains one ormore corneal endothelial cells can be obtained from stem cells. Theobtained cell population containing one or more corneal endothelialcells may be immediately sorted to obtain corneal endothelial cells, ormay be sorted after performing repeated passage for a predeterminedperiod to obtain corneal endothelial cells.

There is no particular limitation to the means for obtaining a cellpopulation containing one or more corneal endothelial cells by culturingcorneal endothelial cells isolated from a cornea, and any method knownor hereafter developed in the art can be suitably selected for use. Forexample, a cell population containing one or more corneal endothelialcells can be obtained in accordance with the method disclosed inWO2014/104366. Specifically, a Descemet's membrane is removed from ahuman corneoscleral tissue with human corneal endothelial cells attachedto the Descemet's membrane, and shredded, followed by culture in amedium containing about 0.2% of collagenase in 5% CO₂ at 37° C. for 1 to3 hours. A usable medium is a DME medium containing 15% fetal calf serum(FCS) and a 2 ng/mL basic fibroblast growth factor (bFGF). Thefibroblasts are then removed by centrifugation wash, followed by trypticdigestion, thereby obtaining a cell population containing pellet-likecorneal endothelial cells (primary culture cells).

The thus-obtained cell population containing one or more cornealendothelial cells may be further cultured in a basal medium typicallyused for animal cell culture, such as D-MEM and MEM. The concentrationof glucose added to the medium is preferably 2.0 g/L or less, and morepreferably 0.1 to 1.0 g/L. It is also preferable to add to the medium agrowth factor such as a hepatocyte growth factor (HGF), epidermal growthfactor (EGF), recombinant EGF (rEGF), and/or fibroblast growth factor(FGF). Of these factors, a single factor or a combination of two or morefactors may be added to the medium. The concentration of the growthfactor(s) in the medium is typically 1 to 100 ng/mL, and preferably 2 to5 ng/mL. From the standpoint of efficient culture of corneal endothelialcells, it is preferable to add a 5 to 1,000 μg/mL ascorbic acidderivative, such as ascorbic acid 2-phosphate, to the medium.

The culture of corneal endothelial cells can be performed by adherentculture using a culture vessel (e.g., a dish, a Petri dish, a tissueculture dish, a multidish, a microplate, a microwell plate, amultiplate, a multiwell plate, a chamber slide, a Schale, a tube, atray, and a cell culture bag) coated with a matrix such as matrigel orcollagen. The culture temperature is typically 35 to 38° C., andpreferably 37° C. The humidity is typically 90 to 100% humidity, andpreferably 100% humidity. The CO₂ concentration is typically 5 to 15%,and preferably 10%. It is thus preferable to culture the cells in anincubator capable of maintaining the conditions. The time period forculture is not particularly limited, and, for example, cells can becultured until the stage at which the cells become confluent (steadystate) (e.g., 1 to 5 days).

The cells can optionally be further passaged, after becoming confluent.For example, confluent cells are washed with PBS, and then dispersedusing trypsin/EDTA, followed by centrifugation. The cells are thenseeded onto a culture vessel containing the same medium as describedabove at a cell density of 500 to 60,000 cell/cm², and cultured underthe conditions described above. Additionally, the cells can be furtherrepeatedly passaged in the same manner after becoming confluent. Thisprocedure provides a cell population containing corneal endothelialcells that are passage cells.

1-2. Sorting Corneal Endothelial Cells Using Molecular Marker asIndication

In a corneal endothelial cell, the following are specifically expressed:ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, CLRN1, SCNN1D, PKD1, CNTN6, NSF,CNTN3, PPIP5K1, and/or PCDHB7. Thus, it is possible to produce one ormore corneal endothelial cells by sorting out one or more cornealendothelial cells from a cell population containing corneal endothelialcells using the expression of at least one molecule selected from thesemolecules as an indication. In this specification, ZP4, MRGPRX3, GRIP1,GLP1R, HTR1D, CLRN1, SCNN1D, PKD1, CNTN6, NSF, CNTN3, PPIP5K1, andPCDHB7 are also referred to as molecular markers.

ZP4 is a gene that encodes one of the glycoproteins that form the zonapellucida, which is an extracellular matrix surrounding the oocyte(Hartmann J F, et al., (1972), Proc Natl Acad Sci USA, 69: 2767-2769).Although the greatest portion of the protein resides outside the cell,its carboxyl terminus is a cellular transmembrane domain (Gupta S K, etal. (2012), Cell Tissue Res, 349: 665-678).

MRGPRX3 is a gene that encodes one of the Mas-related G-protein-coupledreceptors, and is thought to be involved in modulation of pain insensory neurons (Lembo P M, et al. (2002), Nat Neurosci 5: 201-209).

GRIP1 is a gene that encodes a protein that binds to the carboxylterminus of the intracellular domain of an AMPA(a-amino-3-hydroxy-5-methyl-4-isoxazole propionate) glutamic acidreceptor, and is reported as being expressed in the synapses and brain(Dong H, et al., (1999), J Neurosci, 19: 6930-6941).

GLP1R is a gene that encodes a receptor of glucagon-like peptide-1,which is one of the incretins that stimulate insulin secretion from βcells in the pancreas (Orskov C, (1992), Diabetologia, 35: 701-711).GLP1R is reported as being expressed in the pancreas (Tornehave D, etal., (2008), J Histochem Cytochem, 56: 841-851).

HTR1D is a gene that encodes one of the serotonin receptors, and isexpressed in nerve fibers of the craniofacial tissue. HTR1D is thoughtto be involved in the development of migraine (Harriott A M, et al.,(2008), Cephalalgia, 28: 933-944).

CLRN1 is reported as one of the causative genes for Usher syndrome typeIIIa, which causes inner ear disorders and retinitis pigmentosa (KremerH, et al., (2006), Hum Mol Genet 15 Spec No 2: p. 262-270). CLRN1 isexpressed in hair cells in the inner ear and glial cells in the retina,supposedly playing a crucial role in the development and differentiationof hair cells in the inner ear (Geller S F, et al., (2009), PLoS Genet5: e1000607). However, its exact function remains unknown.

SCNN1D is a gene that encodes the 5 subunit of the epithelial Na channel(ENaC), and is reported as being expressed in organs, such as brain,heart, respiratory organ, and kidney (Ji H L et al., (2012), Am JPhysiol Lung Cell Mol Physiol. 303: 1013-1026).

PKD1 is a gene that encodes polycystin-1, and is reported as being oneof the causative genes of autosomal dominant polycystic kidney (Gabow PA, (1993) N Engl J Med. 329: 332-342). PKD1 is highly expressed in thekidney. Polycystin-1 is suggested as being involved in differentiationof renal tubular epithelial cells (Boletta A et al., (2003) Trends CellBiol. 13: 484-492).

CNTN6 is a gene that encodes contactin 6. Contactin 6, which is a memberof the immunoglobulin superfamily, functions as a cell adhesionmolecule. Contactin 6 is suggested as being involved in the formation ofsynaptic connections during nervous system development(http://www.ncbi.nlm.nih.gov/gene/27255).

NSF is a gene that encodes an N-ethylmaleimide-sensitive factor. Thisprotein forms a conjugate together with SNAP (soluble NSF attachmentprotein) and SNARE (SNAP receptor) to stimulate the fusion of thesynaptic vesicle with the cellular membrane (Sollner T et al., (1993)Nature. 362: 318-324).

CNTN3 is a gene that encodes contactin 3. The structure, as withcontactin 6, suggests its role in cell adhesion. CNTN3 is reported asbeing expressed in the frontal lobe, occipital lobe, cerebellum, etc.(http://www.uniprot.org/uniprot/Q9P232).

PPIP5K1 is a gene that encodes an inositol kinase, and is thought toplay a critical role in intracellular signal transduction (Gokhale N Aet al., (2013) Biochem J. 453: 413-426).

PCDHB7 is a gene that encodes a protocadherin β7. Although its specificfunction is unknown, PCDHB7 is suggested as playing a critical role incell-cell adhesion in the nervous system(http://www.ncbi.nlm.nih.gov/gene/56129).

The molecular marker used as an indication for corneal endothelial cellsmay be only one member selected from the group consisting of ZP4,MRGPRX3, GRIP1, GLP1R, HTR1D, CLRN1, SCNN1D, PKD1, CNTN6, NSF, CNTN3,PPIP5K1, and PCDHB7, or the marker may be any combination of two or moreof these molecules. In an embodiment, the molecular marker is preferablyZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, and CLRN1, more preferably ZP4,MRGPRX3, GRIP1, GLP1R, and HTR1D, still more preferably ZP4, MRGPRX3,GRIP1, and GLP1R, still yet more preferably ZP4, MRGPRX3, and GRIP1,still further more preferably ZP4, and MRGPRX3, and particularlypreferably ZP4, because their expression products are surface proteinsof corneal endothelial cells and they are expressed highly specificallyin corneal endothelial cells.

SCNN1D, PKD1, CNTN6, NSF, CNTN3, PPIP5K1, and PCDHB7 are thought to bealso expressed in other tissues, such as the brain, spinal cord,skeletal muscle, or liver, as shown in FIG. 1. However, their expressionin these tissues can be ignored by adjusting the type of stem cells fromwhich a cell group containing one or more corneal endothelial cells isderived and/or the conditions under which differentiation of stem cellsinto corneal endothelial cells are performed, or by using a cell groupobtained by culturing corneal endothelial cells as a cell groupcontaining one or more corneal endothelial cells. Thus, these molecularmarkers are also useful in sorting or detecting corneal endothelialcells. Of these molecular markers, SCNN1D, PKD1, CNTN6, NSF, CNTN3, andPPIP5K1 are preferable, SCNN1D, PKD1, CNTN6, NSF, and CNTN3 are morepreferable, SCNN1D, PKD1, CNTN6, and NSF are still more preferable,SCNN1D, PKD1, and CNTN6 are still yet more preferable, SCNN1D and PKD1are still further more preferable, and SCNN1D is particularlypreferable.

Sorting of one or more corneal endothelial cells from a cell populationcontaining one or more corneal endothelial cells using, as anindication, the expression of at least one molecular marker selectedfrom the group consisting of ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, CLRN1,SCNN1D, PKD1, CNTN6, NSF, CNTN3, PPIP5K1, and PCDHB7 can be performed,for example, by sorting cells in which at least one of the molecularmarkers is expressed, in the case where the cell population containscorneal endothelial cells but does not contain other cells that allowthe expression of the at least one molecular marker. When the cellpopulation contains other cells that allow the expression of the atleast one molecular marker in addition to corneal endothelial cells, thecells that exhibit significantly higher expression levels than othercells can be sorted to isolate corneal endothelial cells. The molecularmarker for use as an indication may be any combination of two or more,three or more, four or more, or five or more of the molecules.

The expression of the molecular marker can be detected by any availablemethod. Examples of typical detection methods include a methodcomprising measuring the expression of the gene(s) that encode the atleast one molecular marker by RT-PCR, and a method comprising detectingthe presence of the at least one molecular marker using a substance thatspecifically binds to the at least one molecular marker. All of themolecular markers listed above are a surface protein of the cornealendothelial cell. Because the molecular marker can be detected withcells remaining viable, the method comprising detecting a molecularmarker using a substance that specifically binds to the marker ispreferable.

The type of substance that specifically binds to the molecular marker isnot particularly limited. Examples of the substance include antibodiesand aptamers. The substance is preferably an antibody or its fragment.The antibody may be either a polyclonal antibody or a monoclonalantibody. Examples of the antibody fragment include Fab fragments,F(ab)₂ fragments, and ScFv fragments. When expression of two or moretypes of molecular markers is used to detect corneal endothelial cells,substances that specifically bind to respective markers may be used incombination. In an embodiment, the substances that specifically bind torespective molecular markers are preferably those that cannot bedecomposed by a treatment to separate the cell group containing one ormore corneal endothelial cells into individual cells (e.g., treatmentwith a protease).

The antibody for specifically recognizing a molecular marker for use maybe a commercially available antibody, or may be prepared by a well-knownmethod. There are well-known methods for preparing antibodies. Forexample, a polyclonal antibody can be prepared by immunizing a non-humananimal with purified molecular markers or their partial peptides, andobtaining serum of the animal in accordance with an ordinary method. Amonoclonal antibody can be obtained from a hybridoma prepared by fusingspleen cells from an immunized animal with myeloma cells.

To facilitate the detection of cells bound to a substance thatspecifically binds to a molecular marker, the substance is preferablylabeled with a labeling substance. The labeling substance is notparticularly limited. Examples of labeling substances includefluorescent materials, radioactive materials, chemiluminescentmaterials, enzymes, biotin, and streptavidin. The substance thatspecifically binds to a molecular marker may be indirectly labeled. Forexample, a pre-labeled antibody (a secondary antibody) that specificallybinds to the substance (e.g., an antibody) may be used.

The method for recognizing cells bound to a substance that specificallybinds to a molecular marker to sort the cells is not particularlylimited, and any method known and hereafter developed may suitably beselected. For example, the method for recognizing/sorting cells can beselected according to the type of labeling substance for use. Typicalmethods for recognizing/sorting cells include fluorescence-activatedcell sorting (FACS), magnetic-activated cell sorting (MACS), andaffinity chromatography. Because of their capability of simultaneouslydetecting multiple molecular markers, FACS and MACS are preferable, andFACS using a flow cytometer equipped with a cell sorter is morepreferable. The mode of FACS is not particularly limited. For example,FACS may be either a droplet-charge mode or a cell-capturing mode.Corneal endothelial cells can be purified in this manner.

As described above, one or more corneal endothelial cells can beobtained by sorting corneal endothelial cells from a cell populationcontaining one or more corneal endothelial cells using a molecularmarker as an indication. The one or more corneal endothelial cellsinclude corneal endothelium precursor cells. Following the methoddescribed above, it is also possible to obtain a cell populationcontaining corneal endothelial cells at a significantly highconcentration. Thus, the method for producing one or more cornealendothelial cells described above can also be used to purify (enrich)corneal endothelial cells. The percentage of purified (concentrated)corneal endothelial cells in a cell population, on a cell count basis,is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70%or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% ormore, or 99% or more. The corneal endothelial cells and cornealendothelium precursor cells (a cell population containing concentratedcorneal endothelial cells and/or corneal endothelium precursor cells)obtained by the method described above can be further cultured. Thefurther culture can be performed, for example, in adifferentiation-induction medium so that the corneal endothelial cellsbecome more mature. Examples of such a differentiation-induction mediumfor use include the above-described medium for inducing differentiationof stem cells into corneal endothelial cells. The further culture can beperformed under conditions suitable for proliferation of cornealendothelial cells, or maintenance of the viability of the cells.

1-3. Use of Corneal Endothelial Cells

The corneal endothelial cells obtained by using the molecular markerdescribed above as an indication can be used in the treatment ofdiseases caused by functional disorder of the corneal endothelium. Thus,there can be provided a pharmaceutical composition containing thecorneal endothelial cells for treatment of corneal endothelial diseases.This pharmaceutical composition may further contain various components,scaffold materials, carriers, etc., to aid the maintenance andproliferation of the corneal endothelial cells, or to aid application ofthe composition to the affected area.

Examples of the components to aid the maintenance and/or proliferationof the cells include components used for mediums, such as carbonsources, nitrogen sources, vitamins, minerals, salts, and variouscytokines. Examples of the scaffold materials to aid the application ofthe composition to the affected area include collagen, polylactic acid,hyaluronic acid, cellulose, and derivatives thereof. These componentsand scaffold materials may be used in a combination of two or more. Thepharmaceutical composition may be in the form of an injectable aqueoussolution (e.g., physiological saline; physiological buffers, such asPBS; and isotonic solutions containing glucose and/or other adjuvants)to which corneal endothelial cells have been added. In one embodiment,the pharmaceutical composition containing the corneal endothelial cellsis preferably in the form of a suspension in which corneal endothelialcells are homogeneously suspended. In clinical applications, such asuspension has been injected into an eye. The concentration of cornealendothelial cells in the suspension is not particularly limited, and is,for example, 1×10⁴/ml to 1×10⁸/ml, or 1×10⁵/ml to 1×10⁷/ml.

A corneal endothelial cell sheet can be obtained by culturing cornealendothelial cells obtained using the molecular marker as an indicationon a suitable carrier (e.g., a polymer membrane). The carrier is notparticularly limited, and examples of the carrier include biopolymers,such as collagen, atelocollagen, alkali-treated collagen, gelatin,keratin, hyaluronic acid, glycosaminoglycan (chondroitin sulfate,dermatan sulfate, hyaluronic acid, heparan sulfate, heparin, keratansulfate), proteoglycan, alginic acid, chitosan, polyamino acid(polylactic acid), and cellulose; and temperature-responsive polymers,such as (meth)acrylamide compounds, N-(or N,N-di)alkyl substituted(meth)acrylamide derivatives, vinyl ether derivatives, and copolymersthereof. In addition to the sheet form, the corneal endothelial cellsmay be used in the form of pellets obtained by concentrating the cells,for example, by filter filtration (a mass of cells).

A protectant or similar component may optionally be added to the cornealendothelial cells to cryopreserve the cells. Examples of the protectantinclude glycerol, DMSO (dimethyl sulfoxide), propylene glycol, andacetamide. To maintain the safety of the cells as a graft, the cells canbe subjected to heat treatment and/or radiation treatment, or the like.

There is no particular limitation to the diseases targeted by thetreatment using corneal endothelial cells, a pharmaceutical compositioncontaining the cells, or a corneal endothelial cell sheet. Examples ofthe diseases include corneal endothelial dysfunction including bullouskeratopathy, corneal dystrophy, developmental glaucoma, Rieger'sanomaly, congenital hereditary endothelial dystrophy, limbal dermoid,sclerocornea, corneal shape irregularities, such as keratoconus andpellucid marginal corneal degeneration, corneal scarring, cornealinfiltration, corneal deposits, corneal edema, corneal ulcer, ocularinjuries caused by a chemical substance or heat, ocular diseases such askeratitis, corneal degeneration, corneal infection, neuroblastoma,Hirschsprung's disease, Waardenburg syndrome, partial albinism, and vonRecklinghausen's disease.

A patient in need of transplant of corneal endothelial cells (e.g., apatient with any of the diseases as described above) can be transplantedwith corneal endothelial cells or a corneal endothelial cell sheet totreat their disease. Thus, a method can be provided for treating thediseases listed above, and the method comprises administering thecorneal endothelial cells obtained in the method described above to apatient in need of transplant of corneal endothelial cells.

2. Method for Evaluating Corneal Endothelial Cells

The at least one member selected from the group consisting of ZP4,MRGPRX3, GRIP1, GLP1R, HTR1D, CLRN1, SCNN1D, PKD1, CNTN6, NSF, CNTN3,PPIP5K1, and PCDHB7 described above is specifically expressed in cornealendothelial cells. Thus, there can be provided a method for evaluatingcorneal endothelial cells using expression of the at least one molecularmarker as an indication. For example, the at least one molecular markeris measured for their expression level in test cells considered (orpresumed) to be corneal endothelial cells. When specific expression (orsignificant expression) is confirmed, the test cells are determined tobe corneal endothelial cells. Preferable molecular markers for use inthis evaluation method are the same as those described in section 1-2above.

There is no particular limitation to the test cells. Examples of testcells include cells obtained by culturing the cells described in section1-1 above in a medium under conditions suitable for differentiationinduction into corneal endothelial cells, and cells obtained byculturing corneal endothelial cells collected from a cornea. The methodfor measuring the expression level of a molecular marker is notparticularly limited. Examples of the method include the measurementmethod using RT-PCR and the measurement method using a substance thatspecifically binds to a molecular marker described in section 1-2. Testcells evaluated in this manner can be safely used as corneal endothelialcells.

3. Corneal Endothelial Cell Detection Kit

The invention provides a kit for detecting corneal endothelial cells,and the kit includes a substance that specifically recognizes at leastone molecular marker selected from the group consisting of ZP4, MRGPRX3,GRIP1, GLP1R, HTR1D, CLRN1, SCNN1D, PKD1, CNTN6, NSF, CNTN3, PPIP5K1,and PCDHB7. The substance that specifically recognizes at least onemolecular marker is not particularly limited. Examples of the substanceinclude the antibodies and aptamers described in section 1-2 above. Thekit may include, in addition to the substance that specificallyrecognizes a molecular marker, any substance used in detection ofcorneal endothelial cells, a container, a manual guide, etc. Forexample, the kit may include a labeling substance that labels thesubstance that specifically recognizes a molecular marker describedabove.

Examples

The following Examples describe the present invention in more detail.However, the invention is not limited to the Examples.

1. Identification of Candidate Molecules

RNA-seq data (GSE41616, Chen, et al., (2013), Hum Mol Genet 22:1271-1279) reported by Chen et al. were obtained from a gene expressioninformation database (Gene Expression Omnibus: GEO) to use asinformation of genes expressed in corneal endothelial cells in humans invivo. The data include information of RNAs expressed in cornealendothelial cells obtained from three adult donors (31, 56, and 64 yearsold), and information of RNAs expressed in corneal endothelial cellsobtained from two fetal donors (16 to 18 weeks). To confirm the dataintegrity, the information of corneal epithelial cell specific markersKRT3 and KRT12 were examined, and high expression levels of KRT3 andKRT12 in the 56-year-old adult donor-derived corneal endothelial cellswas confirmed. Because the sample derived from this donor was likelycontaminated with corneal epithelial cells, data of the remaining fourdonors were used for the following analysis.

RNA-seq reads were aligned to the human reference genome (hg19) by usingTopHat (version 1.4.1), and the results were used to assemble transcriptmodels by Cufflinks package (version 2.1.1) (Trapnell, et al., (2012),Nat. Protoc. 7: 562-578). The gene expression levels were quantified asFPKM (Fragments Per Kilobase of exon per Million mapped fragments: FPKM)using Cufflinks, and genes expressed in corneal endothelial cells at 10FPKM or more were selected; more specifically, 10,627 genes wereselected. Subsequently, these genes were narrowed down to only genesencoding cellular membrane proteins using GO terms listed in Table 1. Asa result, the genes were narrowed down to 1494 genes, as shown inTable 1. Of these, 1075 genes were expressed in both adult and fetalcorneal endothelial cells, 225 genes were expressed in only adultcorneal endothelial cells, and 194 genes were expressed in only fetalcorneal endothelial cells.

TABLE 1 GO Term GO ID Both Adult Fetal Plasma Membrane GO: 0005886 762155 124 Integral to Plasma Membrane GO: 0005887 215 49 57 Cell SurfaceGO: 0009986 111 20 28 Apical Plasma Membrane GO: 0016324 59 18 9Basolateral Plasma Membrane GO: 0016323 50 12 8 External Side of PlasmaGO: 0009897 31 9 9 Membrane Lateral Plasma Membrane GO: 0016328 10 0 4Basal Plasma Membrane GO: 0009925 8 2 2 Extrinsic to Plasma Membrane GO:0019897 8 2 0 Apicolateral Plasma Membrane GO: 0016327 5 0 1 Anchored toExternal Side of GO: 0031362 4 0 0 Plasma Membrane Anchored to PlasmaMembrane GO: 0046658 2 2 0 Extrinsic to External Side of GO: 0031232 2 00 Plasma Membrane Intrinsic to External Side of GO: 0031233 1 1 0 PlasmaMembrane Intrinsic to Plasma Membrane GO: 0031226 1 1 0 Cell OuterMembrane GO: 0009279 1 0 0 External Side of Cell Outer GO: 0031240 0 0 0Membrane Integral to Cell Outer GO: 0045203 0 0 0 Membrane Total 1,075225 194

Finally, genes that expressed at 10 TPM or more (tags per million) in 5or more tissues or cell species were discarded on the basis of theFANTOM5 (Functional Annotation of the Mammalian Genome 5) database. Thisnarrowed down genes encoding candidate molecules to the 13 genes shownin Table 2 below.

TABLE 2 FANTOM5 CAGE RNA-seq Primary FPKM Value Samples Adult PrimarySample Express Gene CEC Fetal CEC Expresses Highest (tpm) >10 tpm GenesHighly expressed in only adult CECs PPIP5K1 17.39 6.17 none 0 CLRN114.15 0.56 lens epithelial cells 2 (22.91) MRGPRX3 11.16 0.26Malassez-derived cells 1 (26.14) GLP1R 10.85 2.64 fetal heart (10.51) 1Genes Highly expressed in only fetal CECs CNTN3 5.36 19.86 none 0 PCDHB71.11 11.14 dura mater (9.37) 0 HTR1D 7.41 10.48 small intestine (12) 2Genes Highly expressed in both adult and fetal CECs GRIP1 39.33 22.70fetal temporal lobe (6.46) 0 NSF 31.58 14.09 pineal gland (7.84) 0 PKD124.99 38.47 aorta (8.45) 0 SCNN1D 21.72 28.05 granulocyte macrophage 4progenitor (22.21) ZP4 12.22 56.52 none 0 CNTN6 11.48 22.09 cerebellum(24.20) 42. RNA Expression Levels of Candidate Molecules in Ocular Tissues andWhole Body Tissues

To confirm the expression levels of the 13 genes narrowed down insection 1 above in corneal endothelial cells and the whole body tissues,quantitative PCR was performed to examine their RNA expression levels inadult human corneal endothelial cells and 22 other tissues. The detailsof the procedure will be described later. The results reveal that all ofthe genes were expressed in corneal endothelial cells, which isconsistent with the results of the data analysis as shown in FIG. 1. Inparticular, ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, and CLRN1 exhibited thehighest expression levels in corneal endothelial cells than in othertissues, while their expression was also confirmed in only a few othertissues.

Subsequently, quantitative PCR was performed on these 6 genes to confirmtheir expression levels across ocular tissues. To prepare oculartissues, four eyeballs from two adult donors were used. As shown in FIG.2, all of the genes were confirmed to exhibit high expression levels incorneal endothelial cells. Given that the corneal stroma is adjacent tothe corneal endothelium and originates from the cranial neural crest aswith corneal endothelial cells, the absence of expression or lowerexpression levels in the corneal stroma is considered to be one of theimportant features for a corneal endothelial cell marker. CLRN1 wasexpressed at a substantially low level in the corneal stroma, and theother 5 genes were not expressed in the corneal stroma. The resultsindicate that all of these molecules are useful as markers specific tocorneal endothelial cells.

Extraction of RNA from corneal endothelial cells was performed inaccordance with the following procedure. All human samples were handledaccording to the tenets of the Declaration of Helsinki. Research-gradecorneoscleral rim and whole eyeballs from a human donor were procuredfrom Sight Life (Seattle, Wash.). The corneoscleral tissues wereimmersed and preserved in Optisol-GS (Bausch & Lomb, Rochester, N.Y.) at4° C., and used within 4 days from the death of the donor. The age ofthe donor was 58 years old. The corneoscleral rim was washed withphosphate-buffered saline (PBS) three times, and the corneal endotheliumand Descemet's membrane were dissected along Schwalbe's line withtweezers. From the dissected corneal endothelium, RNA was extracted witha Qiagen miRNeasy Mini Kit (QIAGEN Inc.).

Extraction of RNA from cultured corneal endothelial cells was performedin accordance with the following procedure. Corneoscleral rims wereobtained from 4 eyeballs from 4 donors. The ages of the donors rangedfrom 14 to 25 years. The corneal endothelia and Descemet's membraneswere isolated as described above. The isolated tissues were incubated ina Dulbecco's modified Eagle medium (DMEM: Invitrogen) containing 1.2U/mL dispase II (Godo Shusei Co., Ltd.) and 1% Antibiotic-Antimycotic(Anti-Anti; Invitrogen/Gibco) at 37° C. for 1 hour to isolate thecorneal endothelial cells from the Descemet's membranes. The isolatedcorneal endothelial cells were gently centrifuged and colleted, and thensuspended in a DMEM medium containing 50 U/mL penicillin, 50 μg/mLstreptomycin, 10% fetal bovine serum (ICN Biomedicals, Inc., Aurora,Ohio), and 2 ng/mL basic fibroblast growth factor (bFGF; Invitrogen).The cells were seeded on dishes coated with a cell attachment reagent(FNC coating mix; Athena ES, Baltimore, Md.), and incubated at 37° C. ina humidified atmosphere of 5% CO₂. RNA was then extracted using anIsogen RNA extraction kit. All the cells used for RNA extraction werethose harvested during the first passage.

Extraction of RNA from human ocular tissues was performed in accordancewith the following procedure. Four eyeballs from 2 donors were preservedin a moist chamber at 4° C., and were used within 5 days from the deathof the donors. The ages of the donors were 75 and 79 years old. First,corneoscleral tissues were prepared, and the ciliary body, iris, andlens were isolated from the anterior segment of each eye, and then theiris stroma and iris pigment epithelial cells were isolated. The cornealendothelium and Descemet's membrane were peeled in the manner describedabove, and the trabecular meshwork was isolated. Further, theconjunctiva was dissected from the corneoscleral tissue, and the centralcornea part and limbal part separated with an 8.0-mm diameter trephinewere treated with Dispase I (Godo Shusei Co., Ltd., Tokyo) at 4° C.overnight. The corneal epithelium and limbal epithelium were separatedfrom the stroma. The neural retina was peeled from the posterior segmentof each eye with tweezers, and the retina pigment epithelial cells (RPE)and choroid were removed together as a cluster. Finally, the optic nervewas isolated. RNAs of all these isolated tissues were extracted using anISOGEN RNA extraction kit.

For RNA samples from the whole human body tissues except for oculartissues, Human Total RNA Master Panel II (#636643; Clontech, MountainView, Calif.) was purchased. Because this product did not include kidneyRNA and pancreas RNA, Human Kidney Total RNA (#AM7976; Ambion, Austin,Tex.) and Human Pancreas Total RNA (#AM7954; Ambion) were alsoseparately purchased.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)was performed in accordance with the following procedure. cDNA wasreverse-transcripted from each RNA with a SuperScript III First-StrandSynthesis System (Invitrogen, Carlsbad, Calif.), and quantitativereal-time PCR (an initial cycle at 95° C. for 30 sec, followed by 45cycles of 95° C. for 5 sec, 60° C. for 30 sec, and 72° C. for 30 sec)was performed to quantify cDNA. SYBR Premix Dimer Eraser (Takara BioInc., Shiga, Japan) was used, and the internal control used was β-actin(ACTB). Table 3 shows the list of primers used.

TABLE 3 Forward/ Accession Gene Reverse Primer Base Sequence SEQ No.Number ACTB Forward 5′-ACAGAGCCTCGCCTTTGC-3′ SEQ NO. 1 NM_001101 Reverse5′-GCGGCGATATCATCATCC-3′ SEQ NO. 2 GRIP1 Forward5′-ATGTGGACAAGAAGCAGCAC-3′ SEQ NO. 3 NM_021150 Reverse5′-GGAGTTTTGGCAACTTCGAC-3′ SEQ NO. 4 NSF Forward5′-CCTATTGGCCCTCGATTTTC-3′ SEQ NO. 5 NM_006178 Reverse5′-GGCTAGTGGTCCCAATGATAAG-3′ SEQ NO. 6 PKD1 Forward5′-AAGACACCCACATGGAAACG-3′ SEQ NO. 7 NM_001009944 Reverse5′-CCAGCGTCTCTGTCTTCTCC-3′ SEQ NO. 8 SCNN1D Forward5′-TGGAGCTGCTACACAACACC-3′ SEQ NO. 9 NM_001130413 Reverse5′-GAGCAGGTCTCCACCATCAG-3′ SEQ NO. 10 ZP4 Forward5′-AAACAGGCCCTCAGGGGA-3′ SEQ NO. 11 NM_021186 Reverse5′-GACAGGTCACCACACAGGAT-3′ SEQ NO. 12 CNTN6 Forward5′-TTCTGAGTCGGAAGGCAAAG-3′ SEQ NO. 13 NM_014461 Reverse5′-CGGACAGATACTGTGCTTCTTG-3′ SEQ NO. 14 PPIP5K1 Forward5′-CTTTCCCTACGTCAAGTGAGTG-3′ SEQ NO. 15 NM_014659 Reverse5′-GCTGCTGTGCATGGAATC-3′ SEQ NO. 16 CLRN1 Forward5′-AATGCAGTACGGGCTTTTCC-3′ SEQ NO. 17 NM_174878 Reverse5′-GCTCACTGGGATTGCTTTG-3′ SEQ NO. 18 MRGPRX3 Forward5′-GGAGGTCTTCACCACTGGAC-3′ SEQ NO. 19 NM_054031 Reverse5′-ACCCAAGACTGGGATGGTTG-3′ SEQ NO. 20 GLP1R Forward5′-GCAGAAATGGCGAGAATACC-3′ SEQ NO. 21 NM_002062 Reverse5′-TTCATCGAAGGTCGGTTG-3′ SEQ NO. 22 CNTN3 Forward5′-CCATGGAAACAGTTGATCCTG-3′ SEQ NO. 23 NM_020872 Reverse5′-GCTGTTGCTGGGTTCTTTG-3′ SEQ NO. 24 PCDHB7 Forward5′-GATTTTGTGCGGTCGCTCTAC-3′ SEQ NO. 25 NM_013940 Reverse5′-TCCCCATTACTTCCGGTATC-3′ SEQ NO. 26 HTR1D Forward5′-CATGCGTTTCTTCCACTGAG-3′ SEQ NO. 27 NM_000864 Reverse5′-CATCGGCACTGCAAATACTG-3′ SEQ NO. 283. Immunostaining of Corneal Endothelial Cells in Corneal Tissue

To confirm the expression of the 6 genes in protein level, cornealtissue sections from a human donor were immunostained using theantibodies shown in Table 4 below. As shown in FIG. 3, in every proteinstaining, the corneal endothelium was intensely stained. In particular,the three antibodies anti-ZP4 antibody, anti-GLP antibody, andanti-HTR1D antibody specifically stained only the corneal endothelium.Although the corneal stroma was also stained by GRIP1, CLRN1, andMRGPRX3, the fluorescence intensity was clearly lower than the intensityin the corneal endothelial cells. It is thus possible to discriminatecorneal endothelial cells from a corneal stroma on the basis of thissignificant difference in expression level. As noted above, the 6 genesand proteins encoded by the 6 genes were found to be useful as markersto specifically recognize corneal endothelial cells.

TABLE 4 Dilution Antibody Source Species and Type Used GRIP1 glutamatereceptor Abcam, Cambridge, MA Rabbit Polyclonal 1:100 interactingprotein 1 Cat Antibody No. ab122514 ZP4 zona pellucida LifeSpanBioSciences, Rabbit Polyclonal 1:50  glycoprotein 4 Cat No. LS- Inc.Seattle, WA Antibody C160968 CLRN1 clarin 1 Cat. No. sc- Santa Cruz GoatPolyclonal 1:50  69073 Biotechnology, Inc. Antibody Santa Cruz, CAMRGPRX3 MAS-related GPR, Abcam, Cambridge, MA Rabbit Polyclonal 1:25 member X3 Cat No. ab140863 Antibody GLP1R glucagon-like peptide GenetexInc., Irvine, Rabbit Polyclonal 1:100 1 receptor CA Antibody Cat No.GTX44806 HTR1D 5-hydroxytryptamine Abcam, Cambridge, MA RabbitPolyclonal 1:150 receptor 1D Antibody Cat No. ab140486

The immunostaining was performed in accordance with the followingprocedure. Corneoscleral tissues were used within 11 days after thedeath of the donor. The age of the donor was 27 years old. Thecorneoscleral rim was embedded in optimal cutting temperature (OCT)compound, and frozen sections were cut using a microtome-cryostat(HM560, Thermo Fisher Scientific Inc., Walldorf, Germany) into pieces of10 μm. After drying at room temperature for 30 minutes, the tissuesections were washed with Tris-buffered saline (TBS; Takara Bio Inc.) 3times, and incubated with TBS containing 5% donkey serum and 0.3% TritonX-100 for 1 hour to block non-specific reactions. The sections were thenincubated with respective primary antibodies listed in Table 4 at 4° C.overnight. Subsequently, the sections were again washed with TBS 3times, and incubated with a 1:200 dilution of their respective AlexaFluor 488-conjugated secondary antibodies (Life Technologies) and a1:100 dilution of Hoechst 33342 (#B2261, Sigma-Aldrich) at roomtemperature for 2 hours. The sections were observed with a fluorescentmicroscope (Axio Observer Dl; Carl Zeiss Jena GmbH, Jena, Germany).

4. Expression of ZP4 in Corneal Endothelial Cells Induced from Human iPSCells

Corneal endothelial cells were induced from human iPS cells (providedfrom the Center for iPS Cell Research and Application, KyotoUniversity), which are pluripotent stem cells. As shown in FIG. 4, theexpression level of ZP4 was increased by induction. The expression ofZP4 was measured with RT-PCR. The expression of ZP4 specific to cornealendothelial cells and the precursor cells indicates that ZP4 is a usefulmarker to produce corneal endothelial cells and evaluate cornealendothelial cells.

SEQUENCE TABLE

PCT_corneal endothelial cell marker_20150903_144718_4.txt

The invention claimed is:
 1. A method for producing a cornealendothelial cell, the method comprising: (a) inducing cornealendothelial cell differentiation in a population of stem cells, and (b)screening differentiated cells in (a) for expression of at least onecell marker selected from the group consisting of Zona pellucidasperm-binding protein 4 (ZP4), Mas-related G-protein coupled receptormember X3 (MRGPRX3), glucagon-like peptide-1 receptor (GLP1R),5-hydroxytryptamine receptor 1D (HTR1D), and Clarin-1 (CLRN1), wherein adifferentiated cell expressing at least one of the cell markers is acorneal endothelial cell.
 2. The method according to claim 1, whereinthe screening comprises adding to the differentiated cells of (a) anantibody that specifically recognizes at least one of the cell markers,under conditions permitting binding of the antibody to one of themarkers.
 3. A method for identifying a corneal endothelial cell in apopulation of cells obtained by inducing differentiation of stem cellsinto corneal endothelial cells, the method comprising identifying a cellexpressing at least two cell markers selected from the group consistingof ZP4, MRGPRX3, Glutamate receptor-interacting protein 1 (GRIP1),GLP1R, HTR1D, and CLRN1 in a population of cells.
 4. The methodaccording to claim 1, wherein the corneal endothelial cell expresses atleast two of the markers.
 5. The method according to claim 1, whereinthe corneal endothelial cell expresses at least three of the markers.